Project Abstract: Babesiosis is a zoonosis and an important blood-borne human parasitic infection. This disease has recently gained much attention because of its growing infection rate in humans by transfer from animal reservoirs; because it represents a potential threat to the blood supply; because asymptomatic infections in man are common and these can be life threatening in certain recipients. Despite the impending danger of the spread of this blood-borne infection, and the significant threat that it presents to the safety of blood transfusions, its study has been neglected. There are 4 major sub-populations of asexual parasites defined by their DNA loads (1 N; 2 N; 4N and ?4N iRBC populations). B. divergens appears to regulate the time spent in the host RBC by building an appropriate mix of subpopulations of iRBCs that provides a high flexibility in terms of developmental options based on the proliferative needs of the population. It has become increasingly clear that most cells communicate within their immediate environment by the formation and release of extracellular vesicles (ECVs). Extracellular vesicles derived from ``donor parasitized cells'' can have profound effects on ``recipient cells'' by altering gene expression and modulating signaling pathways resulting in developmental changes. We have identified extracellular vesicles (ECVs) that are released from babesia iRBCs and hypothesize that these ECVs are the mode of communication between infected RCS, leading to the choice of a specific developmental pathway. We will explore this hypothesis by (1) characterizing the repertoire of ECVs released by B. divergens under various physiological conditions and (2) establish whether specific populations of ECVs function in cell-cell communication. By focusing on those intended for extracellular trafficking, we may identify means of intercellular communication that will be analyzed in future studies. Success is predicted by our experience with Babesia biology, robust preliminary data, cutting edge technology and having all necessary reagents in place to perform the studies. This R21 grant will provide an important dataset to the parasite community and provide further evidence of the role of ECVs in parasite pathogenesis.